We propose to continue and extend ongoing studies of the biosynthesis of polyketide, terpenoid, and other microbial metabolites. Among the natural products to be examined are the macrolide antibiotics methymycin (1), nargenicin (2), and erythromycin (3) and the sesquiterpene pentalenolactone (4) and its parent hydrocarbon pentalenene (5). We also plan to use a combination of recombinant and enzymological techniques to elucidate the final steps in the assembly of pyridoxol (11), the precursor of vitamin B6 (6). The focus of these studies will be on the key steps in the overall biosynthetic pathways with the emphasis on the elucidation of bond-making, bond-breaking, and bond-conservation processes, based on the application of a variety of state-of-the-art precursor-incorporation, enzymatic, molecular genetic, and recombinant methodologies. It is expected that the information gained from such studies will be broadly applicable to the understanding of polyketide, terpenoid, and other natural product biosynthetic processes in general. 1) Studies of the polyketide chain-building reactions in the biosynthesis of three biogenetically closely related macrolide antibiotics, methymycin (1), nargenicin (2), and erythromycin (3) will be extended by the examination of the oxidative metabolism of 10-deoxymethynolide (7), the parent aglycone of methymycin; by the synthesis and incorporation of pentaketide and octaketide precursors into nargenicin (8); and by the testing of rationally designed mutants and the use of cell-free extracts to elucidate the individual biochemical steps in the formation of the reduced polyketide chain of erythromycin. 2) The structural gene for the sesquiterpene cyclase, pentalenene synthase, a Streptomyces enzyme which catalyzes the cyclization of farnesyl diphosphate (8) to pentalenene (5), the parent hydrocarbon of the pentalenolactone family of antibiotics will be expressed in both E. coli and Streptomyces lividans. The overexpressed recombinant cyclase will be used for a battery of experiments involving substrate analogs, active-site directed reagents, and site-directed mutagenesis, designed to elucidate further the mechanism of the cyclization reaction and to reveal the nature of the active site of the synthase itself. 3) The final steps in the construction of the characteristic pyridoxine ring of vitamin B6 will be investigated in collaboration with Prof. Ian Spenser of McMaster University. PCR will be used to amplify the pdxA and pdxJ genes previously implicated in the conversion of D - 1 -deoxyxylulose (9) and 4hydroxythreonine (10) to pyridoxol (11). The PCR products will be inserted into suitable E. coli expression vectors and the resulting overexpressed proteins will be used by Prof. Spenser and his collaborators to examine the formation of pyridoxol.